Glia re‐sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Abstract

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and b-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-aM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca 2+ ionophore ionomycin (0. The role of glia in the brain is an area of intense investigation. In the past decade, exciting results in this field have led to dramatic conceptual changes about the role of glial cells, which were formerly thought to provide only structural and trophic support to neurones. An increasing number of papers have suggested that glia share at least some of the features typical of neurones, particularly those concerned with excitatory neurotransmission (for a review see Haydon 2001). In fact, glial cells are

P2X7 pre‐synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP‐induced glutamate release

“…Purified preparations of rat brain nerve terminals (synaptosomes) ensure very low contamination of non-neuronal cells (Nakamura et al 1993;Lundy et al 2002; see also Gualix et al 2003). Rat cerebrocortical synaptosomal (and gliosomal) fractions had been characterized in our labs (Stigliani et al 2006); here, we confirm that the synaptosomal fraction was contaminated to a negligible extent: purified synaptosomes, positive for the neuronal marker synaptophysin, were negative for the glial marker GFAP, the microglia marker integrin-aM or the oligodendrocyte marker RIP. Release monitoring from a superfused synaptosomal monolayer (Raiteri et al 1974), by minimizing metabolism and removing any released compound prevents indirect effects, avoiding the receptor biophase and enabling 'nude' receptors to be exposed; in these experimental conditions, only targets located on glutamatergic nerve terminals (when monitoring glutamate release) are selectively acted upon, allowing a detailed pharmacological characterization of release-regulating pre-synaptic receptors.…”

“…After decapitation, the cerebral cortex was rapidly removed and placed in ice-cold medium, and purified synaptosomes were prepared as previously reported (Nakamura et al 1993;Stigliani et al 2006). Briefly, the tissue was homogenized in 10 volumes of 0.32 mol/L sucrose, buffered at pH 7.4 with Tris-HCl, using a glass-Teflon tissue grinder (clearance 0.25 mm).…”

Mechanisms of glutamate release from astrocytes

“…These vesicles were shown to be predominantly clear and heterogeneous in size, ranging from 30 to over 100 nm. Furthermore, the presence of clear smooth and clathrin-coated vesicles with apparent diameters of ~30 nm has been observed in gliosomes, a purified preparation of re-sealed fragments of astrocytes from the adult rat brain (Stigliani et al, 2006), which expressed synaptobrevin 2 and VGLUT 1.…”

Glycinergic nerve endings in hippocampus and spinal cord release glycine by different mechanisms in response to identical depolarizing stimuli

“…3 H]glycine release from hippocampal synaptosomes which consisted, however, of crude preparations contaminated by gliosomes, GLYT1-rich astroglial particles (Stigliani et al 2006). A detailed knowledge of glycine release and its regulation in the hippocampus is important particularly because the amino acid can determine the level of activity of NMDA receptors whose glycinebinding sites are not saturated under physiological conditions.…”